Agents that bind to and inhibit human cytochrome P450 2C8, 2C9, 2C18 and 2C19

ABSTRACT

The invention provides monoclonal antibodies to human cytochrome P450 2C8, 2C9, 2C18, and 2C19 having advantageous properties, including capacity substantially to inhibit enzyme activity of the various human cytochrome P450 2C family members and lack of specific binding to other human cytochromes P450. The binding agents of the invention are useful inter alia in methods for screening drugs for metabolism by cytochrome P450 2C family members, and in methods of screening individuals for a poor metabolizing individual human P450 2C family phenotypes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/119,972 filed Feb. 12, 1999, the disclosure of which is incorporatedby reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT TECHNICAL FIELD

The present invention resides in the technical fields of immunology andenzymology.

BACKGROUND OF THE INVENTION

The cytochrome P450 family of enzymes is primarily responsible for themetabolism of xenobiotics such as drugs, carcinogens and environmentalchemicals, as well as several classes of endobiotics such as steroidsand prostaglandins. Members of the cytochrome P450 family are present invarying levels and their expression and activities are controlled byvariables such as chemical environment, sex, developmental stage,nutrition and age.

More than 200 cytochrome P450 genes have been identified. There aremultiple forms of these P450 and each of the individual forms exhibitdegrees of specificity towards individual chemicals in the above classesof compounds. In some cases, a substrate, whether it be drug orcarcinogen, is metabolized by more then one of the cytochromes P450.

Cytochrome P450 2C series of enzyme are of major importance in the useof drugs for the treatment of various disease conditions. The 2C familyranks among the most important of all of the P450 enzymes in humans.

Human cytochrome P450 2C9 is of major importance being responsible forthe metabolism of numerous drugs and non-drug xenobiotics includingtolbutamide, S-warfarin, phenytoin, diclofenac, ibuprofen, and losarten.The three major alleles of P450 2C9 are the wild type 2C9_(Arg144)(*1),2C9_(Cys144)(*2), 2C9_(Ile→Leu359)(*3) (Haining et al. 1996, Miners andBirkett 1998; hereinafter referred to as 2C9*1, 2C9*2 and 2C9*3respectively). A genetic study of the 2C9 polymorphism indicated thatthe frequency of the 2C9* 1 and 2C9*3 alleles in a Caucasian AmericanPopulation sere 0.08% and 0.06% respectively and 0.005 and 0.01%respectively in Afro-Americans (Sullivan-Klose et al. 1996). In aJapanese population the 2C9*2 allele was absent and the frequency of2C9*3 was 0.021 (Nasu et al. 1997). In another study of 100 Caucasiansthe allelic frequency for the 2C9 wild type, 2C9*1, 2C9*2 and 2C9*3 were0.79, 0.12 and 0.085 respectively (Stubbins et al. 1996).

The catalytic roles of the 2C9*1, 2C9*2 and 2C9*3 for the metabolism ofwarfarmn, flurbuprofen and diclofenac were studied in liver microsomesfrom 30 humans that were genotyped for the three 2C9 alleles. Nineteenof the humans were wild type, eight were heterozygous 2C9*2 and threewere heterozygous for 2C9*3. All of the individuals with the 2C9*2allele had similar but slightly lower activities for the metabolism ofthe three substrates. One of the three samples from heterozygotes for2C9*3 had low V_(max) and high K_(m) while the other two samples werecomparable to that of the 2C9*1 wild type and the 2C9*2. The conclusionof the study was that the 2C9*2 allele exhibited comparable but slightlylower metabolic activity than the wild type and the 2C9*3 had slowerrates of oxidation compared to the wild type (Yamazaki et al. 1998). Inanother study (Veronese et al. 1991) found that the V_(max) for the2C9*2 allele for tolbutamide and phenytoin metabolism were 2-3 lowerthan the wild type 2C9. In a different study the V_(max) of (S)7-hydroxywarfarmn formation was 20-fold greater in the wild type than the 2C9*2.The V_(max) for methyihydroxy tolbutamide formation however was similarin the wild type and the 2C9*2 allelic (Rettie et al. 1994).

Typical substrates for 2C family members include taxol, tobutamide,pheytoin, lansoprazolem mephytoin, arachidonic acid, cyclophosphamide,ifosphamide, debrisoquine, methoxylflurane, tienilic acid, phenanthrenetolbutamide, benzo(a)pyrene, 58C80(2-(4-t-Butylcyclohexhl)-3-hydroxyl-1,4-naphthoquinone), torsemide,aracidonic acid, mephenytoin, 1-tetrahydrocannabinol, and warfarin.

Genetic polymorphisms of cytochromes P450 result inphenotypically-distinct subpopulations that differ in their ability toperform biotransformations of particular drugs and other chemicalcompounds. These phenotypic distinctions have important implications forselection of drugs. For example, a drug that is safe when administeredto most humans may cause toxic side-effects in an individual sufferingfrom a defect in an enzyme required for detoxification of the drug.Alternatively, a drug that is effective in most humans may beineffective in a particular subpopulation because of lack of a enzymerequired for conversion of the drug to a metabolically active form.Further, individuals lacking a biotransformation enzyme are oftensusceptible to cancers from environmental chemicals due to inability todetoxify the chemicals. Eichelbaum et al., Toxicology Letters 64/65,155-122 (1992). Accordingly, it is important to identify individuals whoare deficient in a particular P450 enzyme, so that drugs known orsuspected of being metabolized by the enzyme are not used, or used onlywith special precautions (e.g., reduced dosage, close monitoring) insuch individuals. Identification of such individuals may indicate thatsuch individuals be monitored for the onset of cancers.

Existing methods of identifying deficiencies in patients are notentirely satisfactory. Patient metabolic profiles are often assessedwith a bioassay after a probe drug administration. Poor metabolizers(PM) exhibit physiologic accumulation of unmodified drug and have a highmetabolic ratio of probe drug to metabolite. This bioassay has a numberof limitations: Lack of patient cooperation, adverse reactions to probedrugs, and inaccuracy due to coadministration of other pharmacologicalagents or disease effects. See, e.g., Gonzalez et al., Clin.Pharmacokin. 26, 59-70 (1994). Genetic assays by RFLP (restrictionfragment length polymorphism), ASO PCR (allele specific oligonucleotidehybridization to PCR products or PCR using mutant/wild-type specificoligo primers), SSCP (single stranded conformation polymorphism) andTGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE(mutation detection electrophoresis) are time-consuming, technicallydemanding and limited in the number of gene mutation sites that can betested at one time.

A complication in patient drug choice is that most drugs have not beencharacterized for their metabolism by P450 2C family and othercytochromes P450. Without knowing which cytochrome(s) P450 is/areresponsible for metabolizing an individual drug, an assessment cannot bemade for the adequacy of a patient's P450 profile. For such drugs, thereis a risk of adverse effects if the drugs are administered to poormetabolizers.

Monoclonal antibodies that specifically bind to 2C family members andinhibit its activity, if available, could be used to screen drugs fortheir metabolism by 2C and/or identify 2C poor metabolizers by simplebioassays, thereby overcoming the problems in prior complicated methodsdiscussed above. However, such monoclonal antibodies represent, at best,a small subset of the total repertoire of antibodies to human cytochromeP450 2C, and have not hitherto been isolated. Although in polyclonalsera, many classes of antibody may contribute to inhibition of enzymeactivity of P450 2C family members as a result of multiple antibodies insera binding to the same molecule of enzyme, only a small percentage ofthese, if any, can inhibit as a monoclonal. A monoclonal antibody caninhibit only by binding in such a manner that it alone block orotherwise perturb the active site of an enzyme. The existence andrepresentation of monoclonal antibodies with inhibitory properties thusdepend on many unpredictable factors. Among them are the size of theactive site in an enzyme, whether the active site is immunogenic, andwhether there are any sites distil to the active site that can exertinhibition due to stearic effects of antibody binding. The only means ofobtaining antibodies with inhibitory properties is to screen largenumbers of hybridoma until one either isolates the desired antibody orabandons the task through failure.

Notwithstanding these difficulties, the present invention provides interalia monoclonal antibodies that specifically bind to human cytochromeP450 2C family members and inhibit their activity.

SUMMARY OF THE INVENTION

The invention provides isolated binding agents that compete with amonoclonal antibody MAb 292-2-3 for specific binding to human cytochromeP450 allelic variant 2C9*2 without specifically binding to humancytochrome 2C9*1 and 2C9*3, and that specifically inhibits 2C-catalyzedmetabolism of phenanthrene by at least 50%. The invention furtherprovides other isolated binding agents that compete with a monoclonalantibody MAb 763-15-5 for specific binding to the human cytochrome p4502C9 allelic variants 2C9*1, 2C9*2, and 2C9*3, and that specificallyinhibits 2C-catalyzed metabolism of phenanthrene by at least 50%. Theinvention further provides isolated binding agents that compete with amonoclonal antibody MAb 763-15-20 for specific binding to the humancytochrome P450 2C9 allelic variants 2C9*1, 2C9*2, and 2C9*3. Theinvention further provides isolated binding agents that compete with amonoclonal antibody MAb 592-2-5 for specific binding to human cytochromeP450 2C9 and 2C18, and that specifically inhibits 2C-catalyzedmetabolism of phenanthrene by at least 50%. The invention furtherprovides isolated binding agents that compete with a monoclonal antibodyMAb 5-7-5 for specific binding to a human cytochrome p450 2C familymember selected from the group consisting of 2C9, 2C18, and 2C19, andthat specifically inhibits 2C-catalyzed metabolism of phenanthrene by atleast 50%.

Preferred binding agents are monoclonal antibodies. Some binding agentslack specific binding to at least one cytochrome P450 selected from thegroup consisting of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9,2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Some binding agents lack specificbinding to each of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9, 2C18,2C19, 2D6, 2E1, 3A4, and 3A5. Preferred binding agents are able tospecifically inhibit the enzyme activity of human cytochrome P450allelic variant 2C9*2 by at least 90%. Other preferred binding agentsare able to specifically inhibit the enzyme activity of human cytochromeP450 allelic variant 2C9*2 by at least 70%. Other preferred bindingagents are able to specifically inhibit the enzyme actibity of humancytochrome P450 allelic variants 2C9*1 and 2C9*3 by at least 70%. Somebinding agents are binding fragments, such as Fab fragments.

MAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, MAb 281-1-1, MAb763-15-5, and MAb 763-15-20 are exemplified monoclonal antibodies. Someother monoclonal antibodies are analogs of these monoclonal antibodiescomprising a light chain variable domain having at least 80% sequenceidentity with the light chain variable domain of a monoclonal antibodyselected from the group consisting of MAb 292-2-3, MAb 592-2-5, MAb5-7-5, MAb 5-1-5, MAb 281-1-1, MAb 763-15-5, and MAb 763-15-20, whereinthe percentage sequence identity is determined by aligning amino acidsin the light chain variable domains by the Kabat numbering conventionand a heavy chain variable domain having at least 80% sequence identitywith the heavy chain variable domain of a monoclonal antibody selectedfrom the group, wherein the percentage sequence identity is determinedby aligning amino acids in the heavy chain variable domains by the Kabatnumbering convention.

The invention further provides cell lines producing monoclonalantibodies as described above. Cells lines can be eucaryotic orprocaryotic.

The invention further provides methods of determining whether acytochrome P450 2C family member metabolizes a compound. Such methodsentail contacting the compound with cytochrome P450 2C family member inthe presence of varying amounts of the binding agents above. Metabolismof the compound is then assayed as a function of amount of bindingagent, a decrease of metabolism with amount of binding agent indicatingthat cytochrome P450 2C family member metabolizes the compound. In somesuch methods, the compound is contacted with cytochrome P450 2C familymember in a sample containing a collection of cytochrome P450 enzymesincluding the 2C family member. Preferred P450 2C family members areP450 2C9*1, 2C9*2, 2C9*3, 2C8, 2C18, and 2C19.

In some methods, the sample is a tissue sample. In some methods, thecollection of enzymes are obtained from a cell culture expressing theenzymes. In some methods, the compound is a drug, steroid or carcinogen.

The invention further provides methods of detecting cytochrome P450 2Cmembers. Such methods entail contacting a sample suspected of containingcytochrome P450 2C family member with a binding agent described above.One then determines whether the agent specifically binds to the sample,specific binding indicating the presence of the particular cytochromeP450 2C family member in the sample.

The invention further provides methods of measuring P450 2C levels in anindividual relative to P450 levels in a control population. Such methodsentail contacting a sample suspected of containing cytochrome a P450 2Cfamily member from the individual and a substrate of 2C. One thendetermines the P450 2C levels in the individual relative to P450 2Clevels in the control population.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Immunoblot analysis of expressed human P450s with MAb 592-2-5,MAb 5-7-5, MAb 5-1-5, MAb 281-1-1, and MAb 292-2-3.

FIG. 2 Inhibition of phenanthrene metabolism by monoclonal antibodiesMAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, and MAb 281-1-1 to humanP450 2C family members. The incubation and separation were performed asreferenced (Shou et al., 1994).

FIG. 3 Inhibition of diazepam metabolism by monoclonal antibodies MAb292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, and MAb 281-1-1 to humanP450 2C family members.

FIG. 4 Specificity analysis of MAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb5-1-5, and MAb 281-1-1 to human P450 2C family members. Immunoblots,ELISA, and inhibition results are shown.

FIG. 5. Specificity of Binding of Monoclonal Antibody 292-2-3 toExpressed Human P450 Family Members.

FIG. 6. Specificity of MAb 292-2-3: Inhibition of Metabolism by P450 2C9Alleles and Other P450 Family Members.

FIG. 7. Tolbutamide and Diclofenac Metabolism by 2C9: SpecificInhibition of the P450 2C9_(Cys 144)(*2) Allele by MAb 292-2-3.

FIG. 8. Specificity of Binding of MAb 763-15-5 and MAb 763-15-20 toExpressed Human P450 Family Members.

FIG. 9. Specificity of MAb 763-15-5: Lack of Inhibition of Other HumanP450 Family Members.

FIG. 10. Immunoblot Analysis of MAb 763-15-5 and MAb 763-15-20.

FIG. 11. Inhibition of P450 2C9 Allele Metabolism of Diclofenac.

DEFINITIONS

Specific binding between an antibody or other binding agent and anantigen means a binding affinity of at least 10⁶ M⁻¹. Preferred bindingagents bind human cytochrome P450 2C family members with affinities ofat least about 10⁷ M⁻¹, and preferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹.Lack of specific binding means a binding affinity of less than 10⁶ M⁻¹.

Lack of inhibition means that P450 metabolism in the presence of anexcess of antibody is inhibited by less than 10% of the value in theabsence of antibody.

The term epitope means a protein determinant capable of specific bindingto an antibody. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a humanized immunoglobulin orthe amino acid sequence of the humanized immunoglobulin) refers to twoor more sequences or subsequences that have at least about 80%, mostpreferably 90-95% or higher nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usingthe following sequence comparison method and/or by visual inspection.Such “substantially identical” sequences are typically considered to behomologous. Preferably, the “substantial identity” exists over a regionof the sequences that is at least about 50 residues in length, morepreferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues, or over the fall length of the two sequences to becompared. As described below, any two antibody sequences can only bealigned in one way, by using the numbering scheme in Kabat. Therefore,for antibodies, percent identity has a unique and well-defined meaning.That is, percent sequence identity is the percentage of aligned aminoacids or nucleotides that are the same between two immunoglobulins ortheir coding sequences being compared.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acids according to thescheme of Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) Kabatet al. list many amino acid sequences for antibodies for each subclass,and list the most commonly occurring amino acid for each residueposition in that subclass. Kabat et al. use a method for assigning aresidue number to each amino acid in a listed sequence, and this methodfor assigning residue numbers has become standard in the field. Kabat etal's scheme is extendible to other antibodies not included in thecompendium by aligning the antibody in question with one of theconsensus sequences in Kabat et al. The use of the Kabat et al.numbering system readily identifies amino acids at equivalent positionsin different antibodies. For example, an amino acid at the L50 positionof a human antibody occupies the equivalence position to an amino acidposition L50 of a mouse antibody.

The term antibody is used to mean whole antibodies and binding fragmentsthereof.

An isolated species means an object species (e.g., a binding polypeptideof the invention) that is the predominant species present (i.e., on amolar basis it is more abundant than any other individual species in thecomposition). Preferably, an isolated species comprises at least about50, 80 or 90 percent (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

DETAILED DESCRIPTION

The invention provides monoclonal antibodies and other binding agents inisolated form that specifically bind to human cytochrome P450 2C8,2C9*1, 2C9*2, 2C9*3, 2C18, and 2C19, and inhibit enzymic activity ofthese human cytochromes. Preferred agents lack specific binding to otherhuman cytochromes P450. The invention further provides methods of usingthe antibodies and other binding agents in identifying individuals witha poor metabolizing 2C family member phenotypes, and in screening drugsfor metabolism by cytochrome P450 2C family members.

I. Binding Agents of the Invention

A. Specificity and Functional Properties

Binding agents of the invention compete with exemplary antibodiesdesignated MAb 292-2-3 (ATCC HB-12645), MAb 592-2-5 (ATCC-12646), MAb5-7-5 (ATCC-12647), MAb 5-1-5 (ATCC HB-12649), MAb 281-1-1 (ATCCHB-12648), MAb 763-15-5 (ATCC PTA-1079), and MAb 763-15-20 (ATCCPTA-1078), for specific binding to human cytochrome P450 2C familymembers. Production of monoclonal antibodies MAb 292-2-3, MAb 592-2-5,MAb 5-7-5, MAb 5-1-5, MAb 281-1-1-1, MAb 763-15-5, and MAb 763-15-20 isdescribed in the Examples.

1. MAb 292-2-3, MAb 763-15-5, and MAb 763-15-20

The data in the Examples show that Mab 292-2-3 specifically binds onlyto the single expressed human P450 2C9_(Cys 144)(*2) and does not bindthe wild type human P450 2C9_(Arg 144)(*1) or the human P450 2C9*3. MAb292-2-3 inhibits the enzyme activity of P450 2C9_(Cys 144)(*2) by morethan 90% as measured with phenanthrene metabolism. MAb 292-2-3 does notinhibit the enzyme activity of the other two P450 2C9 alleles, the P4502C family members 2C8, 2C18, 2C19, or human P450 1A1, 1A2, 2A6, 2B6,2D6, 2E1, 3A4, and 3A5. MAb 292-2-3 does not immunoblot P4502C9_(Cys 144)(*2), 2C9_(Arg 144)(*1), or 2C9*3, 2C8, 2C18, 2C19, humanP450 1A1, 1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5. MAb 292-2-3 identifiesand determines the quantitative amount of a drug substrate metabolizedby P450 2C9_(Cys 144)(*2) and therefore its role in drug metabolism.

MAb 763-15-5 specifically inhibits all three 2C9 allelic variants. MAb763-15-5 specifically inhibits the enzyme activity of P4502C9_(Arg 144)(*1) by more than 85%; specifically inhibits the enzymeactivity of P450 2C9_(Cys 144)(*2) by more than 90%; and specificallyinhibits the enzyme activity of P450 2C9*3 by more than 75%, as measuredby phenanthrene, bufuralol, or diclofenac metabolism. MAb 763-15-5 alsoinhibits human P450 2C18 by 30% as measured by phenanthrene metabolism.MAb 763-15-5 does not immunoblot any of the P450 2C9 alleles, 2C8, 2C18,2C19, human P450 1A1, 1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5.

MAb 763-15-20 immunoblots all three human P450 2C9 alleles as well ashuman P450 2C8. Mab 763-15-20 does not immunoblot P450 2C18, 2C19, 1A1,1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5. Human P450 2C8 can bedistinguished by its large difference in migration relative to the humanP450 2C9 family. At very high concentrations (greater than in normaluse) a slight band is observed with human P450 2C19.

2. Mab 5-1-5 and MAb 281-1-1

MAb 5-1-5 and MAb 281-1-1 specifically inhibit the enzyme activity ofhuman P450 2C8 as measured with phenanthrene, diazepam and taxolmetabolism. MAb 5-1-5 and MAb 281-1-1 do not inhibit the enzyme activityof the other P450 2C family members 2C9, 2C18, 2C19 or human P450 1A1,1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5.

MAbs 281-1-1 and 5-1-5 yield an immunoblot only to the target 2C8 andnot to the other P450 members. The immunoblot data further indicatesthat these antibodies bind to epitope(s) that are not lost on treatmentwith a denaturing solvent. Both MAbs 281-1-1 and 5-1-5 identify drugsubstrates for human P450 2C8, determine the amount of 2C8 proteinpresent in a sample, and its role in drug metabolism.

3. MAb 592-2-5

MAb 592-2-5 specifically forms an immunoblot and inhibits the enzymeactivity of human P450 2C9 and 2C18. These two antibodies do not inhibitthe enzyme activity of the other P450 2C family members 2C8 and 2C19 orhuman P450 1A1, 1A2, 2A6, 2B6, 2D6, 2E1, 3A4, and 3A5.

4. MAb 5-7-5

MAb 5-7-5 inhibits the enzyme activity of 2C9/18/19 but not 2C8 and theother P450. MAb 5-7-5 yields a strong immunoblot with 2C9 and a weak tomoderate immunoblot to 2C8, 2C18, and 2C19. FIG. 4 provides a summary ofresults with the MAbs of the invention to each of the 2C family P450 forELISA, immunoblot and enzyme inhibition studies.

Hybridomas producing MAb 292-2-3 (ATCC HB-12645), MAb 592-2-5 (ATCCHB-12646), MAb 5-7-5 (ATCCHB-12647), MAb 5-1-5 (ATCC-HB 12649), MAb281-1-1 (ATCC HB-12[4]648), MAb 763-15-5 (ATCC PTA-1079), and MAb763-15-20 (ATCC PTA-1078) have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassus, Va. 20110-2209nder the Budapest Treaty and given the Accession Nos. indicated. MAb292-2-3 (ATCC HB-12645), MAb 592-2-5 (ATCC HB-12646), MAb 5-7-5(ATCCHB-12647), MAb 5-1-5 (ATCC HB-12649), MAb 281-1-1 (ATCC HB-12648)were given the Accession Nos. indicated on Feb. 3, 1999. MAb 763-15-5(ATCC PTA-1079), and MAb 763-15-20 (ATCC PTA-1078) were given theAccession Nos. indicated on Dec. 21, 1999. These cell lines will bemaintained at an authorized depository and replaced in the event ofmutation, nonviability or destruction for a period of at least fiveyears after the most recent request for release of a sample was receivedby the depository, for a period of at least thirty years after the dateof the deposit, or during the enforceable life of the related patent,whichever period is longest. All restrictions on the availability to thepublic of these cell lines will be irrevocably removed upon the issuanceof a patent from the application.

Competition is determined by an assay in which the antibody under testinhibits specific binding of a reference antibody to an antigenicdeterminant on human cytochrome P450 2C family members. Numerous typesof competitive binding assays are known for example: (see Harlow andLane, “Antibodies, A Laboratory Manual,” Cold Spring Harbor Press(198)). Typically, such an assay involves the use of a purified humancytochrome P450 2C family member, an unlabelled test antibody and alabeled reference antibody. Competitive inhibition is measured bydetermining the amount of label bound to the human cytochrome P450 2Cfamily member in the presence of the test antibody. Usually the testantibody is present in excess. Antibodies identified by competitionassay (competing antibodies) include antibodies binding to the sameepitope as a reference antibody and antibodies binding to an adjacentepitope sufficiently proximal to the epitope bound by the referenceantibody for steric hindrance to occur. Usually, when a competingantibody is present in excess, it will inhibit specific binding of areference antibody to the human cytochrome P450 2C family member by atleast 10, 25, 50 or 75%.

Binding agents of the invention typically lack specific binding (i.e.,crossreactivity) to human cytochromes P450 other than the individual 2Cfamily members, so that the binding agents can be used to detect humancytochrome P450 2C family members in the presence of other cytochromesP450. For example, binding agents of the invention typically lackspecific binding to one or more of human cytochromes P450 1A1, 1A2, 2A6,2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5 as measured by ELISAand immunoblot. Some binding agents of the invention, including theexemplified monoclonal antibodies of the invention lack specific bindingto some or all of the above human cytochromes P450.

As noted above, binding agents of the invention are characterized bycapacity to inhibit a particular human cytochrome P450 2C familymember-catalyzed metabolism of a substrate known to be metabolized bythe enzyme. The enzyme can be assayed with any of diazepam, taxol, andphenanthrene (see present Examples). Assays can be performed in either amicrosome systems or a reconstituted systems of purified enzymes. Forexample, a suitable microsome system contains 1 mg/ml protein of humanliver microsomes or 1.6 mg protein/ml from human lymphoblast cell lines,together with 0.2 mM substrate in a final volume of 1.0 ml of 100 mMpotassium phosphate buffer, pH 7.5, and 1 mM NADPH. An exemplaryreconstituted system, in place of the microsome system, contains about20-50 nM a purified human P450 2C family member, 40-100 nM cytochromeb5, 100 nM NADPH-P450 reductase, 10 μAg/ml phospholipids and 0.25 mMsodium cholate. Incubations are typically carried out at 37° C. for 30min. Percentage inhibition is defined as 1—(rate of formation metabolicproduct in presence of test antibody/rate of formation of metabolicproduct in presence of control antibody), when antibody is present inexcess. (The control antibody is an antibody lacking specific binding tothe particular human cytochrome P450 2C family member being studied.)Some agents of the invention inhibit metabolic capacity of isolated purecytochrome P450 2C family members on any or all of the above substratesby at least 25%, 50%, 75%, 85%, 90% or 95%.

B. Antibodies of the Invention

1. General Characteristics

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat et al., supra. An alternative structural definition has beenproposed by Chothia et al., J. Mol. Biol. 196, 901-917 (1987); Nature342, 878-883 (1989); and J. Mol. Biol. 186, 651-663 (1989).

2. Production

Antibodies to human cytochrome P450 2C family members can be produced bya variety of means. The production of non-human monoclonal antibodies,e.g., murine or rat, can be accomplished by, for example, immunizing theanimal with a preparation containing purified human cytochrome P450 or afragment thereof. The immunogen can be obtained from a natural source,by peptides synthesis or preferably by recombinant expression.Antibody-producing cells obtained from the immunized animals areimmortalized and screened for the production of an antibody which bindsto human cytochrome P450 or a fragment thereof. See Harlow & Lane,Antibodies, A laboratory Manual (CSHP NY, 1988) (incorporated byreference for all purposes).

A cloned human P450 2C cDNA family member of interest can be insertedinto a baculovirus vector and Hi-five or Spondoptera Frugipedra (Sf9)cells (or other suitable cell line) can be infected with the recombinantbaculovirus to produce the particular 2C family member (e.g., see Buterset al., 1994; Gonzalez et al., 1991b). BALB/c mice can then be immunizedby i.p. injection weekly for 3 weeks with 30 mg of the particular 2Cfamily member of interest protein emulsified in complete Freund'sadjuvant for the first injection, and then with incomplete Freund'sadjuvant for subsequent injections. Three days after the thirdinjection, the mouse serum can be examined by ELISA. The mice aresacrificed and spleens removed. The hybridoma production, screening byELISA and IB of MAbs, and MAb content determination are the samedescribed (Gelboin et al., 1996; Gelboin et al., 1995).

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989) and WO 90/07861 (incorporated by reference for allpurposes).

Human antibodies can be obtained using phage-display methods. See, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outersurfaces. Antibodies are usuallydisplayed as F_(v) or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to humancytochrome P450 or a fragment thereof. Human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as MAb 292-2-3, MAb763-15-5, MAb 763-15-20, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, and MAb281-1-1. Such antibodies are particularly likely to share the usefulfunctional properties of the exemplified antibodies.

3. Antibody Fragments

Antibodies of the invention include intact antibodies and fragments.Typically, these fragments compete with the intact antibody from whichthey were derived for specific binding to a particular human cytochromeP450 2C family member, and bind with an affinity of at least 10^(6,)10^(7,) 10^(8,) 10⁹, or 10¹⁰ M⁻¹. Antibody fragments include separateheavy chains, light chains Fab, Fab′ F(ab′)₂, F_(v), and single chainantibodies comprises a heavy chain variable region linked to a lightchain variable region via a peptide spacer. Fragments can be produced byenzymic or chemical separation of intact immunoglobulins. For example, aF(ab′)₂ fragment can be obtained from an IgG molecule by proteolyticdigestion with pepsin at pH 3.0-3.5 using standard methods such as thosedescribed in Harlow and Lane, supra. Fab fragments may be obtained fromF(ab′)₂ fragments by limited reduction, or from whole antibody bydigestion with papain in the presence of reducing agents. (See id.)Fragments can also be produced by recombinant DNA techniques. Segmentsof nucleic acids encoding selected fragments are produced by digestionof full-length coding sequences with restriction enzymes, or by de novosynthesis. Often fragments are expressed in the form of phage-coatfusion proteins. This manner of expression is advantageous foraffinity-sharpening of antibodies.

4. Recombinant Expression of Antibodies

Nucleic acids encoding light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding antibody chainsare operably linked to control sequences in the expression vector(s)that ensure the expression of antibody chains. Such control sequencesinclude a signal sequence, a promoter, an enhancer, and a transcriptiontermination sequence. Expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosome.

E. coli is one procaryotic host particularly for expressing antibodiesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilus, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication) and regulatory sequences such as alactose promoter system, a tryptophan (trp) promoter system, abeta-lactamase promoter system, or a promoter system from phage lambda.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

Mammalian tissue cell culture can also be used to express and producethe antibodies of the present invention (see Winnacker, From Genes toClones (VCH Publishers, N.Y., 1987). Eukaryotic cells are preferred,because a number of suitable host cell lines capable of secreting intactantibodies have been developed. Preferred suitable host cells forexpressing nucleic acids encoding the immunoglobulins of the inventioninclude: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.(USA) 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); and, TRI cells (Mather et al., Annals N.Y.Acad. Sci. 383:44-46 (1982)); baculovirus cells.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell. Calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation can be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). When heavy and light chainsare cloned on separate expression vectors, the vectors areco-transfected to obtain expression and assembly of intactimmunoglobulins. After introduction of recombinant DNA, cell linesexpressing immunoglobulin products are cell selected. Cell lines capableof stable expression are preferred (i.e., undiminished levels ofexpression after fifty passages of the cell line).

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see generally Scopes, ProteinPurification (Springer-Verlag, N.Y., 1982). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred.

5. Screening for Sequence Analog

Many of the antibodies described above can undergo non-criticalaminoacid substitutions, additions or deletions in both the variable andconstant regions without loss of binding specificity or effectorfunctions, or intolerable reduction of binding affinity (i.e., belowabout 10⁶ M⁻¹) for human cytochrome P450 2C family members. Usually, thelight and heavy chain variable regions of immunoglobulins incorporatingsuch alterations exhibit at least 80, 90 or 95% sequence identity to thecorresponding regions of a reference immunoglobulin from which they werederived, such as MAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5 and MAb281-1-1. Preferred antibody light and heavy chain sequence variants havethe same complementarity determining regions (CDRs) as the correspondingchains from one of the above reference antibodies. Occasionally, amutated immunoglobulin can be selected having the same specificity andincreased affinity compared with a reference immunoglobulin from whichit was derived. Phage-display technology offers powerful techniques forselecting such immunoglobulins. See, e.g., Dower et al., WO 91/17271McCafferty et al., WO 92/01047; Huse, WO 92/06204.

C. Other Binding Agents of the Invention

The invention further provides nonantibody binding agents that competewith one of the exemplified antibodies for binding to particular humancytochrome P450 2C family members. These binding agents includepolypeptides, beta-turn mimetics, polysaccharides, phospholipids,hormones, prostaglandins, steroids, aromatic compounds, heterocycliccompounds, benzodiazepines, oligomeric N-substituted glycines andoligocarbamates. Large combinatorial libraries of the compounds can beconstructed by the encoded synthetic libraries (ESL) method described inAffymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each ofwhich is incorporated by reference for all purposes). Peptide librariescan also be generated by phage display methods. See, e.g., Devlin, WO91/18980. The libraries of compounds are screened for binding to humancytochrome P450 in competition with one of the reference antibodies MAb292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5 and MAb 281-1-1.

II. Human Cytochrome P450 2C Family

The cDNA for the human cytochrome P450 2C family members has beencloned, sequenced, and expressed. Sources of other cytochromes P450(e.g., for use in testing for lack of crossreactivity) are described byNebert, DNA & Cell Biol. 10, 1-14 (1991); Nelson et al.,Pharmacogenetics 6, 1-42 (1996). Insect cells (e.g., SF9) withappropriate vectors, usually derived from baculovirus, are also suitablefor expressing 2C family members and other cytochromes P450. See Luckowet al., Bio/Technology 6:47-55-55 (198); Gonzalez et al., Meth.Enzymol., 206, 93-99 (1991) (incorporated by reference for allpurposes). Other expression systems include yeast (Ellis et al., supra),E. coli (Gillam et al., Archives Biochem. Biophys. 319, 540-550 (1995);vaccinia virus (Gonzalez, Pharmacol. Res. 40, 243 (1989), and humanAHH-1 lymphoblastoid cells (Crespi et al., Carcinogenesis 10, 295-301(1989)).

III. Methods of Use

A. Identifying Compounds Metabolized by Particular 2C Family Members

Binding agents of the invention that inhibit enzymic activity of humancytochrome P450 2C family members can be used to assay whether compoundsare metabolized by 2C family members. Compounds include xenobiotics,such as a currently used and new drugs, carcinogens, pesticides or otherindustrial or environmental chemicals, or any endobiotic, such as asteroid hormone. The assay can indicate not only that a compound ismetabolized by a particular 2C family member but also the contributionof the particular 2C family member to metabolizing the compound relativeto other cytochromes P450 present in microsomes or cell homogenates.

Assays are performed by contacting a compound under test with aparticular human cytochrome P450 2C family member in reaction mixturescontaining varying amounts of a binding agent of the invention. Forexample, two separate reactions may be set up, one in which the bindingagent of the invention is present, and the other, a control in which thebinding agent is absent. The human cytochrome P450 is often present as amicrosomal extract from human or animal cells or cell lines or anextract from cell cultures expressing a collection of recombinant P450sincluding the particular 2C family member. The assay is performed underconditions in which the particular 2C family member is known to beactive on known substrates, such as phenanthrene (see Examples).Metabolism of the compound under test is then followed from thedisappearance of the compound or appearance of a metabolic product ofthe compound as a function of time (e.g., nmol product/sec). See, e.g.,Buters et al., Drug Metab. Dispos. 22, 688 (1994). The metabolism of thecompound is analyzed as a function of the amount of binding agentpresent. If the metabolism quantitatively decreases with amount ofbinding agent, it can be concluded that the particular 2C family membermetabolizes the compound.

The percentage inhibition of a particular 2C family member catalyzedmetabolism of a test compound reflects both the inherent efficiency of abinding agent in blocking the 2C family member activity and thus thecontribution of cytochromes P450 other than the particular 2C familymember in metabolizing the compound. The inherent blocking efficiency ofa binding agent can be determined by measuring inhibition of metabolismin a reaction mixture in which only a particular 2C family member ispresent, or alternatively, in a reaction mixture in which a collectionof cytochromes P450 are present but the substrate is known to bemetabolized only by the particular 2C family member. Comparison of thepercentage inhibition determined in these circumstances with thepercentage inhibition of metabolism of a test substrate when a mixtureof cytochromes P450 are present indicates the relative contributions ofthe particular 2C family member and other enzymes in the mixture tometabolism of the test substrate. For example, if metabolism of acontrol substrate by pure 2C8 is inhibited by a binding agent by 90% andmetabolism of a test substrate by a mixture of cytochromes P450including 2C8 is inhibited 45%, it can be concluded that in the mixture,2C8 contributes about 45/90=50% of metabolizing activity on the testsubstrate. Binding agents having a high degree of inhibition (e.g., atleast about 90%) of a known substrate are particularly effective forquantitative analysis as described above.

Information made available by the above methods can be exploited in anumber of applications. Drugs determined to be processed by individual2C family members should in general not be prescribed to patients withPM phenotypes, or should be prescribed in reduced amounts or with closemonitoring. Particular caution is needed in combination therapiesinvolving two drugs metabolized by the 2C pathways. The information canalso be valuable in drug design and screening. That is drugs can beesigned or screened such that they are metabolized to a significantextent by several P450 enzymes, and are not therefore likely to causeside effects in those deficient in any single enzymes. Recognition thata carcinogen or other environmental toxin is deactivated by a particular2C family member signals that poor metabolizers are at particular riskfrom the carcinogen or compound. Conversely, recognition that acarcinogen or other environmental toxin is activated to harmful form bya 2C family member indicates that poor metabolizers are less prone toharm from exposure to such a compound relative to extensivemetabolizers.

B. Use of Agents for Identifying Drugs That can be Used for DiagnosingPM Phenotype

The binding agents are useful diagnostics to determine a patient'smetabolic profile prior to treatment with a drug known or suspected tobe metabolized by a 2C family member. Patients identified as 2C poormetabolizers can be given alternative therapy, a lower dosage oradditional monitoring to avoid damaging side effects from their PMphenotype. Diagnosis can be performed by two assays, which are describedin turn.

1. Binding Assay

Binding agents of the invention are useful for the quantitativemeasurement of the amount of individual P450 proteins in a sample, whichmay contain multiple forms of other P450 proteins. Binding betweenbinding agent and the particular cytochrome P450 2C family member in thesample can be detected by radioimmunoassay, ELISA or immunoblotting (seeHarlow and Lane, supra). The type of immunoassay can be tailored to theparticular application. In radioimmunoassay, the binding agent of theinvention is typically labeled. In ELISA, the binding agent is typicallyunlabelled and detected using a secondary labeled reagent with affinityfor the binding agent. Immunoblots are particularly useful for screeninga sample with a panel of antibodies to different cytochromes P450.

2. Inhibition Assay

In an inhibition assay, the presence or absence of a particular 2Cfamily member in a tissue sample from a patient is determined from thecapacity of a binding agent of the invention to inhibit metabolism of aknown substrate of the 2C family member by the tissue sample. A seriesof reaction mixtures are prepared each of which contains an aliquot oftissue sample from a patient being diagnosed and a known substrate ofthe particular 2C family member (e.g., phenanthrene). The tissue samplecan be obtained from any tissue in which the particular 2C family memberis normally expressed, such as the liver. The reaction mixtures differin the amount of binding agent of the invention present, and a controlis usually included in which no binding agent is present. The bindingagent used should be one that is known to inhibit the particular 2Cfamily member metabolism. The reaction conditions are such that theknown substrate is metabolized to a detectable extent in the control ifthe tissue sample is from an extensive metabolizer. The rate ofsubstrate metabolism is determined in the reaction mixtures and analyzedas a function of amount of binding agent present. If the tissue sampleis from an extensive metabolizer, extensive metabolic activity should beobserved in the control in which no inhibitor is present, and decreasingactivity should be observed in other reaction mixtures as the amount ofbinding agent is increased. Conversely, if the sample is from a poormetabolizer, only background metabolic activity (attributable mainly tometabolism of the substrate by other cytochromes P450) is observed inthe control, and similar levels of activity are observed in the otherreaction mixtures. The lack of correlation between metabolism ofsubstrate and amount of binding agent present signals that the tissuesample is from a poor metabolizer.

C. Other Uses

The binding agents of the invention can also be used for affinitypurification of particular cytochrome P450 2C family members. The basicprocedure for affinity purification requires only one or two steps andcan yield highly purified milligram quantities of a particularcytochrome P450 2C family member. For example, the binding agent can becovalently bound to Sepharose™, which is made into the form of eithercolumn or a slurry for batch purification. A sample containing aparticular cytochrome P450 2C family member is them passed through thecolumn or slurry and binds to the binding agent-linked Sepharose™. Thenonbound material containing unrelated proteins and cytochromes P450other than the particular 2C family member are thoroughly eluted leavingthe cytochrome P450 2C family member of interest, which can then beeluted and used for a variety of chemical and physical studies. See,e.g., Cheng et al., J Biol. Chem. (1948) 259: 12279-12284.

Monoclonal antibody based immunohistochemical methods can be applied tolocalize and examine the distribution of various cytochrome P450 2Cfamily members after different inducer administration, during variousphysiological states related to nutrition, age, and sex, and indifferent species and tissues. Furthermore, the intracellulardistribution of each cytochrome P450 2C family member can be determinedin a way not possible by standard biochemical methods which generallycannot identify the presence of specific forms of cytochrome P450proteins in isolated tissues and organelles. See, e.g., Gelboin,Pharmacol. Rev. 45: 413-453 (1993); Forkert et al., Res Commun ChemPathol Pharmacol. (1986) 53: 147-57; Forkert et al., Carcinogenesis(1991) 12: 2259-2268; and Forkert, et al., Mol. Pharmacol. (198) 34:736-43; these references and the references cited therein are hereinincorporated by reference.

EXAMPLES Materials and Methods

Hybridoma Production

The P450 2C9*2 was expressed using a vaccinia vector in Hep G2 cells(Battula et al. 1987) or from a baculovirus vector in insect cells(Gonzalez et al. 1991) and extracted according to published methods(Gelboin et al. 1998). The extracted P450 baculovirus expressed P4502C9*2 which was used as the immunogen. The vaccinia expressed P450s wereused for some of the enzyme analysis. Balb/c mice were immunized with 50ug of baculo-expressed P450 for three weeks, the spleen cells isolatedand fused with myeloma cells (NS-1). The resulting hybridomas werescreened for antibody production and binding specificity to singleexpressed human P450 by ELISA as previously described (Gelboin et al.1998). Of 1086 hybridomas screened for antibody production and bindingspecificity one was found to bind specifically to P450 2C9*2. All theother hybridomas yielded MAbs that were either non-binding or notspecific to the target P450. The hybridoma producing the desired MAb292-2-3 was cloned three times, and the MAb was produced in cell cultureor by ascites production in mice as described (Gelboin et al. 1998).Other single clones of the 1086 clones that were screened bound to the2C9 alleles, 2C18 and 2C19, respectively. 830 individual clones werescreened to obtain the 2C8 MAbs (MAbs 281-1-1 and 5-1-5). (See Gelboin,H. V., et al. (1998) Monoclonal Antibodies to Cytochrome P450. InMethods in Molecular Biology: Cytochrome P450 Protocols, edited by I. R.Phillips and E. A. Shephard (New Jersey: Humana Press Inc.), pp.227-237; this reference and all references cited therein are hereinincorporated by reference).

Immunoassays

ELISA was performed on ninety-six well plates which were coated with 1.5picomole of a single P450. MAbs derived from culture fluids or bydiluted ascites fluid were applied and incubated at room temperature for2 hours. After washing, labeled antibody (alkaline phosphatase goat antimouse IgG_(Fc)) was applied for 1 hour. After washing, the wells weredeveloped with the appropriate substrate (p-nitrophenyl phosphatase) andthe absorbance was measured at 405 nm. SDS-PAGE gels and Immunoblots(IB) were performed as previously described (Gelboin et al. 1998).

Immunoinhibition studies were performed using cDNA vaccinia expressedP450 enzymes from Hep G2 cells, Supersomes (Gentest Corp.) and/orBaculosomes (Panvera Corp.). Generally the incubations contained: 30-60μg of ascites protein containing the MAb which was pre-incubated with30-50 pmole of P450 enzyme in 0.5 ml in buffer (100 mM Tris pH 7.5) andincubated at 37*C for 5 minutes. The substrate in 10 μl methanol, NADPH,and additional buffer were added to a final volume of 1.0 ml. Theincubation was for 30 minutes (60 mm. for tolbutamide) and themetabolites were extracted with 8m1 DCM. Phenanthrene (200 uM) was usedfor examining the specificity of inhibition of the target P450s by theMAb (Shou et al. 1994). Phenanthrene metabolism was catalyzed by: 1A1,1A2, 2A6, 2B6, 2C8, 2C9(*1), 2C9(*2), 2C9(*3), 2C18, 2C19, 2E1, 3A4, and3A5. Burfurolol (50 uM) metabolism was used for measuring 2D6 metabolism(Gelboin et al. 1997). Diclofenac (50 uM) (Crespi and Penman 1997) andTolbutamide (200 uM) (Relling et al. 1990) were used for testing thespecificity of the MAb 292-2-3 inhibition of metabolism catalyzed byeach of the three 2C9 allelic variants; 2C9*1, 2C9*2, and 2C9*3.Metabolites formed were separated by HPLC performed using a HewlettPackard Model 1050 Series system equipped with an autosampler, a ternarysolvent delivery system, and a dioarray detector controlled by theHewlett Packard Chemstations software. Metabolite retention times werecompared with authentic standards and metabolite peaks were quantitatedbased on their ratios to internal standards. Control incubationscontained an MAb against hen egg white lysozyme (MAb HyHel-9)(Gelboin etal. 1998). Percent of control metabolism was calculated from thatobserved with the absence and presence of the MAb 292-2-3.

Human Liver Microsomes and cDNA-expressed P450s

Human liver specimens, stored at −80° C. until use, were obtained fromorgan donors after clinical death (The NCI Cooperative Human TissueNetwork, NIH, Bethesda, Md.). Microsomes were prepared as described(Alvares et al., 1970) and microsomal protein (Lowry et al., 1951) andP450 content (Omura and Sato, 1964) were measured according to publishedmethods.

MAbs Inhibition of 2C Activity

Inhibition of P450 catalyzed activity was always performed withsaturating levels of MAbs yielding maximum inhibition. A typical assaycontained MAbs in 5 to 25 μL of ascites with a content at about 400 pmolMAb (IgG). This ascites fluid was preincubated with 25 pmol of aparticular 2C family member or 250 pmol of human liver microsomal P450sin 0.5 mL of 50 mM potassium phosphate buffer (KPi, pH7.4) at 37° C. for5 min. The mixture was diluted with the buffer to a final volume of 1mL. The substrate, i.e., diazepam, tolbutamide, taxol, bufuralol, andphenanthrene, was dissolved in 10 mL of methanol and added (finalconcentration at 200 μM), and the reaction was initiated by the additionof NADPH (1 mM) at 37° C. Anti-lysozyme MAb (HyHel, IgG), with an amountequivalent to the test MAbs, was used as a control for nonspecificinhibition. Reactions were incubated for 30 min and terminated with 1 mLof acetone. Samples were extracted twice with 7 mL dichloromethane andwere dried under N2 and the residue dissolved in mobile phase andimmediately analyzed by reversed phase HPLC. The metabolites formed wereidentified by comparing their retention times with authentic standards.

High Performance Liquid Chromatography

HPLC was performed using a Hewlett-Packard (HP, Rockville, Md., USA)Model HP1050 liquid chromatography system equipped with an HP model 1050autosampler, a ternary solvent delivery system, and amultiple-wavelength or dioarray detector, which are controlled by theHPLC 2D or 3D ChemStation software installed on a Compaq Deskpro 5133personal computer (Compaq Computer Cor., Houston, Tex., USA).

Analysis of diazapam metabolism and phenanthrene metabolism (Shou etal., 1994) were previously described, respectively.

RESULTS

Preparation of MAbs Specific to Human P450 2C8, 2C9, 2C18, and 2C19

P450 2C9*2 was expressed using a vaccinia vector in Hep G2 cells(Battula et al. 1987) or from a baculovirus vector in insect cells(Gonzalez et al. 1991) and extracted according to published methods(Gelboin et al. 1998). The extracted P450 baculovirus expressed P4502C9*2 which was used as the immunogen. The vaccinia expressed P450s wereused for some of the enzyme analysis. Balb/c mice were immunized with 50ug of baculo-expressed P450 for three weeks, the spleen cells isolatedand fused with myeloma cells (NS-1). The resulting hybridomas werescreened for antibody production and binding specificity to singleexpressed human P450 by ELISA as previously described (Gelboin et al.1998). Of 1086 hybridomas screened for antibody production and bindingspecificity one was found to bind specifically to P450 2C9*2. All theother hybridomas yielded MAbs that were either non-binding or notspecific to the target P450. The hybridoma producing the desired MAb292-2-3 was cloned three times, and the MAb was produced in cell cultureor by ascites production in mice as described (Gelboin et al. 1998).

FIG. 1 shows the immunoblot analysis of the specificity of the five MAbsto human P450 2C8, 2C9*2, 2C18, and 2C19. MAbs 292-2-3 did not form animmunoblot with any 2C family members. MAb 281-1-1 yielded an immunoblotonly to the target 2C8 and not to the other cytochrome P450 2C familymembers. MAb 592-2-5 formed an immunoblot with 2C9 and 2C18. MAb 5-7-5yielded strong immunoblots with all three 2C9 alleles and a weak tomoderate immunoblot to 2C8, 2C18, and 2C19.

FIG. 4 shows the specificity analysis of MAb 292-2-3, MAb 592-2-5, MAb5-7-5, MAb 5-1-5, and MAb 281-1-1 to human P450 2C family members.Immunoblots, ELISA, and inhibition results are shown. MAb 292-2-3 showsa positive ELISA for 2C9*1, 2C9*2, and 2C9*3 while MAb 592-2-5 shows apositive ELISA for 2C9*2 and a weak ELISA for 2C18. MAb 5-7-5 shows apositive ELISA for 2C9*1, 2C9*2, and 2C9*3, 2C18 and 2C19 while both MAb5-1-5 and MAb 281-1-1 show a moderate ELISA for 2C8.

The immunobinding and ELISA data of MAb 281-1-1, MAb 592-2-5, 5-1-5, andMAb 5-7-5 show that these MAbs are highly specific to 2C8, or 2C9*1,2C9*2, and 2C9*3, 2C18, and 2C19.

Inhibitory Activity of MAb 292-2-3, Mab 5-1-5, MAb 592-2-5, MAb 281-1-1,and MAb 5-7-5 Toward 2C8, 2C9*2, 2C18, and 2C19 Enzyme Activity

The metabolism of two substrates of the 2C family members, phenanthrene(FIG. 2) (Shou et al., 1994) or diazepam (FIG. 3)were used to measurethe inhibitory activity of the MAbs. The data show the inhibitoryactivity of the MAbs toward the 2C family-catalyzed metabolism. Theinhibition of phenanthrene metabolism ranged from 82-96%. The inhibitionof diazepam metabolism ranged from 66-93%. The five MAbs shown are thussensitive and precise probes for measuring 2C-catalyzed metabolism inliver and other tissues.

Specificity of Binding of MAb 292-2-3 to Expressed Human P450 2C9Alleles

The specificity of binding of the MAb 292-2-3 to the expressed P450family members determined by ELISA is shown in FIG. 5. The MAb 292-2-3binds only to the single expressed 2C9*2 and does not bind the wild type2C9*1 or the 2C9*3. Further, the MAb 292-2-3 does not bind to othermembers of the 2C family, 2C8, 2C18, 2C19 and none of the non-2Cisoforms of human liver P450s, i.e., 1A1, 1A2, 2A6, 2B6, 2D6, 2E1 or3A4/5. The MAb 292-2-3 does not immunoblot any of the above three 2C9alleles.

FIG. 6 shows the specificity of inhibition of MAb 292-2-3 for themetabolism of phenanthrene catalyzed by the 2C9*1 and 2C9*2 alleles andother P450 isoforms in human liver except for 2D6 measured by bufuralolmetabolism and 2C9*3 measured by diclofenac metabolism. The metabolismby 2C9*2 was inhibited by at least 90% by MAb 292-2-3 which exhibited nosignificant inhibition of the two other 2C9 alleles, 2C isoforms (8, 18,19) or the non 2C /P450 isoforms, 1A1, 1A2, 2A6, 2B6, 2C8, 2D6, 2E1, 3A4or 3A5. All of the measurements of enzyme activity and inhibition by theMAbs were performed with P450s expressed from either vaccinia orbaculovirus vectors.

The specificity of MAb 292-2-3 toward each of the three 2C9 allelecatalyzed metabolism of two major 2C9 substrates, tolbutamide anddiclofenac, is shown in FIG. 7. The MAb 292-2-3 inhibited 2C9*2 by morethan 95% and did not exhibit significant inhibition of the P450 2C9*1 or2C9*3.

Specificity of Binding of MAb 763-15-5 and MAb 763-15-20 to ExpressedHuman P450 2C9 Alleles

The specificity of binding of MAb 763-15-5 and MAb 763-15-20 to theexpressed human P450 family members by ELIZA is shown in FIG. 8. FIG. 9shows the specificity of MAb 763-15-5. MAb 763-15-5 inhibits each of thethree 2C9 alleles catalyzed metabolism of one of the major 2C9substrates. Phenanthrene metabolism was used for all P405s exceptdiclofenac metabolism for the P450 2C9 alleles and bufuralol metabolismfor P450 2D6. MAb 763-15-5 inhibited 2C9*2 by more than 90% andinhibited the P450 2C9*1 and 2C9*3 by more than 75%. MAb 763-15-20inhibited 2C9*2 by more than 10% and did not exhibit significantinhibition of the P450 2C9*1 or 2C9*3.

The immunoblot analysis of MAb 763-15-5 and MAb 763-15-20 is shown inFIG. 10 MAb 763-15-5 did not yield an immunoblot whereas MAb 763-15-20bound strongly to human P450 2C8 and all three allelic variants of humanP450 2C9.

FIG. 11 shows the inhibition of P450 2C9 allele metabolism of diclofenacby MAb 763-15-5 and MAb 763-15-20. MAb 763-15-5 inhibited 2C9*2metabolism of diclofenac by m ore than 90% and inhibited the P450 2C9*1and 2C9*3 by more than 75%. MAb 763-15-20 inhibited 2C9*2 metabolism ofdiclofenac by more than 10% and did not exhibit significant inhibitionof the P450 2C9*1 or 2C9*3.

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Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

What is claimed is:
 1. A monoclonal antibody that competes with themonoclonal antibody MAb 281-1-1 (ATCC HB-12648) for specific binding tohuman cytochrome P450 2C8, and that specifically inhibits 2C8-catalyzedmetabolism of phenanthrene by at least 50%.
 2. The monoclonal antibodyof claim 1 that lacks specific binding to each of human cytochromes P4501A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5.
 3. The monoclonalantibody of claim 1 that specifically inhibits 2C8-catalyzed metabolismof phenanthrene by at least 90%.
 4. The monoclonal antibody of claim 1that is a mouse antibody.
 5. The monoclonal antibody of claim 1, whereinthe light chain variable domain comprises three complementaritydetermining regions (CDRs) from the light chain of the monoclonalantibody MAb 281-1-1 (ATCC HB-12648), and the heavy chain variabledomain comprises three complementarity determining regions (CDRS) fromthe heavy chain of the monoclonal antibody MAb 281-1-1-1 (ATCCHB-12648).
 6. The monoclonal antibody of claim 1 that is MAb 281-1-1(ATCC HB-12648) or a binding fragment thereof.
 7. A cell line producingthe monoclonal antibody of claim
 6. 8. The cell line of claim 7 that isa eucaryotic cell line.
 9. The cell line of claim 7 that is aprocaryotic cell line.